ABSTRACT Breast cancer is the second leading cause of cancer-deaths in women in the United States and early detection can help improve survival. While mammography is the current first-line imaging technique for early breast cancer detection, its diagnostic accuracy is limited in women with dense breast tissue. Ultrasound is often performed as a second line test in women with dense breast tissue. However, due to its low specificity, it results in many false positive findings with unnecessary biopsies with increased associated health care costs. Dr. Willmann and his group at Stanford have recently identified and validated B7-H3 as a novel, highly specific neoangiogenesis marker in patients with breast cancer that allows differentiation of breast cancer from benign breast lesions with an AUC of up to 0.96. In a preliminary study, they tested an ultrasound contrast agent bearing antibodies to B7-H3 and showed in a transgenic mouse model that contrast-enhanced ultrasound could detect small foci of breast cancer. They have identified a peptide that binds to both murine and human B7-H3. Dr. Unger and his group at NuvOx Pharma developed the world?s #1 selling microbubble (MB) ultrasound contrast agent and have industry-leading expertise in targeting MB technologies. The overall goal of this grant proposal is to develop a novel clinical grade B7-H3-targeted ultrasound contrast agent that allows detection and characterization of breast cancer with both high sensitivity and specificity. In this collaborative effort we will create bioconjugates of the already identified peptide to B7-H3, incorporate them into newly designed clinical grade ultrasound contrast microbubbles (MB), confirm their binding to B7-H3 in vitro under flow shear stress conditions, and then confirm efficacy in a transgenic mouse model of breast cancer development. We will label the molecularly targeted MB with a fluorescent dye to allow in situ confirmation of in vivo B7-H3 target binding using microscopy. Successful completion of the Aims of this project may subsequently support formal development of B7-H3-targeted MB as a new drug candidate in Phase II.